Real-time assay for the detection of botulinum toxin

ABSTRACT

A real-time portable and rapid detection assay to identify the presence of biologically active toxins such as botulinum toxins. The proteolytic activity of BoNT/A is measured using a peptide cleavage assay, where a fluorescent substrate is cleaved by BoNT/A, resulting in increased fluorescence. This fluorescence can be monitored in real-time using a fluorescence detection instrument, such as a real-time PCR system that has been modified to implement a detection algorithm specific to the identification of the target toxin.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/732,436, filed on Dec. 3, 2012.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the detection of biologically activebotulinum toxins and, more specifically, to a real-time assay fordetecting biologically active botulinum toxins that can be implementedin the field.

2. Description of the Related Art

Botulinum neurotoxins (BoNTs) are proteins produced by the bacteriaClostridium botulinum. BoNTs are powerful toxins that cause the lifethreatening illness, botulism, in humans, with BoNT serotype A (BoNT/A)being one of the most potent. BoNTs produce their toxic effects byentering neurons and then cleaving N-ethylmaleimide-sensitive factoractivating protein receptor (SNARE) proteins. In particular, BoNT/Aspecifically cleaves SNAP-25 which prevents the formation of a synapticfusion complex and thereby inhibits the release of acetylcholine,resulting in muscle fiber paralysis. BoNT exposure is fatal withoutimmediate diagnosis and proper treatment. Due to their ease ofproduction, BoNTs pose a major biological warfare threat.

Early detection of BoNTs is crucial for bio-security and food safety.Real-time quantitative polymerase chain reaction (qPCR) is a very commondetection method used in the biodefense field. qPCR is a very sensitiveand quick method for detecting biological organisms by amplifyingspecific regions of deoxyribonucleic acid (DNA), and can be used todetect the genes coding for BoNTs. However, BoNTs are proteins that donot require the intact organism to cause disease, and can be purifiedfrom the organism. The purified toxin, which consists of 100-kDa heavychain (HC; required for cell entry) joined by a disulfide bond to a50-kDa light chain (LC; required for SNAP-25 cleavage), may becompletely devoid of DNA and therefore not detectable using qPCR. qPCRhas the ability to detect the gene coding for a protein toxin, but itdoes not directly detect the presence, or more importantly the activityof protein toxins.

BRIEF SUMMARY OF THE INVENTION

It is therefore a principal object and advantage of the presentinvention to provide a real-time assay for the detection of biologicallyactive botulinum toxins.

In accordance with the foregoing objects and advantages, the presentinvention provides a real-time portable and rapid detection assay toidentify the presence of biologically active toxin such as botulinumtoxins. The detection assay includes a BoNT/A sensing fluorescentsubstrate, a negative control/interferent sensing fluorescent substrate,a qPCR detection protocol modified for toxin identification, and a toxindetection algorithm. The proteolytic activity of BoNT/A can be measuredusing a peptide cleavage assay, where a synthesized dual labeledfluorescent peptide substrate is cleaved by BoNT/A, resulting inincreased fluorescence based on Forester (fluorescence) resonance energytransfer (FRET) principles.

While assays according to the present invention may use a commerciallyavailable fluorescent peptide substrate that mimics the BoNT/A cleavagesite of SNAP-25 (such as SNAPtide® peptide available from ListBiological Laboratories), the detection assay may be made more stableand more sensitive by using a fluorescent peptide substrate (SEQ. IDNO. 1) that mimics both the BoNT/A binding and cleavage sites ofSNAP-25. Additionally, a negative control/interferent sensingfluorescent peptide substrate (SEQ. ID NO. 2) was designed based on SEQ.ID NO. 1 so that it would be cleaved or inhibited by the same proteasesor inhibitors that would affect SEQ. ID NO. 1, but at the same time beinsensitive to BoNT/A proteolytic activity due to a mutated BoNT/Acleavage site.

The increase in fluorescence in SEQ. ID NO. 1 caused by BoNT/A activitycan be monitored in real-time using any temperature controlledfluorimeter (e.g. the FilterMax® F5 Multimode Microplate Readeravailable from Molecular Devices), any lab-based qPCR fluorescencedetection instrument (e.g. the Rotor-Gene® Q available from Qiagen)running a qPCR detection protocol modified for toxin identification, orany field-based qPCR fluorescence detection instrument (e.g. the RAZOR®EX available from BioFire Diagnostics or the Genedrive® available fromEpistem) running a qPCR detection protocol modified for toxinidentification. The ruggedized RAZOR® EX and the small form factorGenedrive® are portable qPCR based platforms designed for use outside ofa laboratory environment that have the ability to detect fluorescencechanges in less than 1 hour for biodefense (RAZOR® EX) and point of carediagnostics (Genedrive®). Fluorescence data generated in thefluorescence detection instruments is then applied to a toxin detectionalgorithm, which utilizes data from a sample exposed to both SEQ. ID NO.1 and SEQ. ID NO. 2 to determine if biologically active BoNT/A toxin ispresent or absent in the test sample. qPCR platforms are preferred overbasic temperature controlled fluorimeters because they allow theoperator to use a single instrument to screen one sample usingconventional qPCR for genetic detection of biological threat agents(such as the Bacillus anthracis and Francisella tularensis qPCR assays)and non-conventional activity screening for biological activitydetection of toxins (such as the BoNT/A activity assay describe here).

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The present invention will be more fully understood and appreciated byreading the following Detailed Description in conjunction with theaccompanying drawings, in which:

FIG. 1 is a schematic of an assay for detecting botulinum toxinaccording to the present invention using the proof-of-principlefluorescent peptide SNAPtide® or the designed fluorescence peptide SEQ.ID NO. 1.

FIG. 2 is a graphical representation of the anticipated fluorescenceresponse and algorithmic data processing of an assay for detectingbiologically active botulinum toxin according to the present invention.

FIG. 3A-3B is a series of graphs displaying results acquired on theRotor-Gene® Q qPCR instrument from the buffer optimization using 10 μMSNAPtide® and 1.9 ng, 3.8 ng, 7.5 ng, and 15 ng BoNT/A-LC.

FIG. 4A-4C is a series of graphs displaying results acquired on theRotor-Gene® Q qPCR instrument using 10 μM SNAPtide® showing the activityof 1.9 ng and 7.5 ng BoNT/A-LC in the presence of ZnCl₂ with varyingconcentrations of DTT and in the presence of the common PCR inhibitorshumic acid or diesel exhaust residue.

FIG. 5 is a graph displaying results acquired on the RAZOR® EX portableqPCR instrument using 10 μM SNAPtide® showing the activity of a range ofBoNT/A-LC amounts (0.11 ng-30 ng) incubated for 55 minutes at 37° C.

FIG. 6 is a graph displaying results acquired on the RAZOR® EX portableqPCR instrument using 10 μM SNAPtide® showing the activity of a range ofBoNT/A-LC (0.06 ng-7.5 ng) using 34.5 mM Hepes buffer pH 7.4+0.03% (v/v)Tween 20+0.31% PBS+0.02% (v/v) Triton X-100.

FIG. 7 is a graph displaying results acquired on the RAZOR® EX portableqPCR instrument using 10 μM SNAPtide® showing the optimization of ZnCl₂and DTT concentrations in the BoNT/A-LC detection assay.

FIG. 8 is a graph displaying results acquired on the RAZOR® EX portableqPCR instrument using 10 μM SNAPtide® showing the detection of 1.9ng-7.5 ng BoNT/A-LC in the presence of the common qPCR inhibitor dieselexhaust residue.

FIG. 9 is a graph displaying results acquired on the RAZOR® EX portableqPCR instrument using 10 μM SNAPtide® showing the detection of 7.5 ngBoNT/A-LC in the presence of the common qPCR inhibitor Arizona roaddust.

FIG. 10 is a graph displaying results acquired on the RAZOR® EX portableqPCR instrument using 10 μM SNAPtide® showing the detection 7.5 ngBoNT/A-LC in the presence of the common qPCR inhibitor humic acid.

FIG. 11A-11D shows three-dimensional graphical representations of theC-terminal region of SNAP-25 binding to BoNT/A-LC which aided in thedesign of a new BoNT/A peptide substrate.

FIG. 12A-12C show the protease cleavage maps of newly designed BoNT/Apeptide substrates.

FIG. 13 is a graph displaying results acquired on the FilterMax® F5fluorimeter using the newly designed SEQ. ID NO. 1 peptide substratecompared to SNAPtide® showing the improvement in detection of variousamounts of BoNT/A-LC.

FIG. 14 is a graph displaying results acquired on the FilterMax® F5fluorimeter showing the requirement of the SNARE domain in SEQ. ID NO. 1(compared to SEQ. ID NO. 6) in the detection of BoNT/A-LC.

FIG. 15A-15B shows graphs displaying results acquired on the FilterMax®F5 fluorimeter showing that only biologically active BoNT/A is detectedby the SEQ. ID NO. 1 BoNT/A peptide substrate.

FIG. 16A-16B shows graphs displaying results acquired on the FilterMax®F5 showing that the SEQ. ID NO. 2 peptide substrate can act as anegative control/interferents sensor for SEQ. ID NO. 1 as it isunresponsive to biologically active BoNT/A-LC, but can detectnon-specific protease activity (such as that found in Arizona RoadDust).

FIG. 17 is a graph displaying results acquired on the FilterMax® F5using SEQ. ID NO. 1 showing the detection of 5.0 ng BoNT/A-LC in thepresence of the common PCR inhibitor diesel exhaust extract.

FIG. 18 is a graph displaying results acquired on the FilterMax® F5using SEQ. ID NO. 1 showing the detection of 2.5 ng BoNT/A-LC in thepresence of the common PCR inhibitor humic acid.

FIG. 19 is a graph displaying results acquired on the FilterMax® F5using SEQ. ID NO. 1 showing the detection of 5.0 ng BoNT/A-LC in thepresence of the reducing reagents TCEP and DTT.

FIG. 20 is a graph displaying results acquired on the FilterMax® F5using SEQ. ID NO. 1 showing the detection of 5.0 ng BoNT A HolotoxinComplex in the presence of varying concentrations of TCEP.

FIG. 21 is a graph displaying results acquired on the RAZOR® EX portableqPCR instrument using SEQ. ID NO. 1 and SEQ. ID NO. 2 showing thedetection of 7.5 ng of BoNT/A-LC.

FIG. 22 is a graph displaying results acquired on the Genedrive®portable qPCR instrument using SEQ. ID NO. 1 showing the detectionvarious amounts of BoNT/A-LC.

FIG. 23 is a graph displaying results acquired on the FilterMax® F5using SEQ. ID NO. 1 showing the limits of detection of BoNT/A-LC.

FIG. 24 is a list of the formulas used in the detection algorithm of thepresent invention.

FIG. 25 is a graph of the raw data of an example according to thedetection algorithm of the present invention.

FIG. 26 is a graph of the adjusted raw data of an example according tothe detection algorithm of the present invention.

FIG. 27 is a graph of a discrete slope comparison according to thedetection algorithm of the present invention.

FIG. 28 is a graph of a discrete slope delta comparison according to thedetection algorithm of the present invention.

FIG. 29 is a graph of a cumulative slope comparison according to thedetection algorithm of the present invention.

FIG. 30 is a graph of a cumulative slope delta comparison according tothe detection algorithm of the present invention.

FIG. 31 is a graph of a slope comparison according to the detectionalgorithm of the present invention.

FIG. 32 is a graph of a slope delta comparison according to thedetection algorithm of the present invention.

FIG. 33 is a graph of a cumulative and discrete adjusted slope accordingto the detection algorithm of the present invention.

FIG. 34 is a graph of a cumulative and discrete mean adjusted slopeaccording to the detection algorithm of the present invention.

FIG. 35 is a graph of the standard deviations according to the detectionalgorithm of the present invention.

FIG. 36 is a graph of the discrete slope standard deviations accordingto the detection algorithm of the present invention.

FIG. 37 is a graph of the cumulative slope standard deviations accordingto the detection algorithm of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, wherein like reference numerals refer tolike parts throughout, there is seen in FIG. 1 a schematic of afunctional protein assay with BoNT/A according to the present invention,where a fluorescently labeled peptide substrate, comprising a peptidesubstrate labeled with a fluorophore and a quencher, such as theFITC/DABCYL labeled proof-of principle substrate SNAPtide® or SEQ. IDNO. 1 (Table 1), is contacted by a sample containing botulinumneurotoxin (BoNT/A). For example, SNAPtide® and SEQ. ID NO. 1 have anN-terminal FITC or internal FAM (fluorophore) tag and a C-terminalDABCYL (quencher) tag. Upon cleavage by BoNT/A-LC, the fluorophore andquencher become spatially separated, resulting in increasedfluorescence. The results may be monitored by an appropriate temperaturecontrolled fluorescence reader, such as a Rotor-Gene® Q (available fromQiagen, Valencia, Calif.), a FilterMax® F5, a Genedrive®, or a RAZOR® EXsystem. As seen in FIG. 2, the increase of fluorescence over time may bemeasured, graphically displayed, and/or compared to a negative controlsample using an algorithm to indicate whether biologically activebotulinum toxin is present in a given sample.

EXAMPLE 1

Materials

The assay according to the present invention was initially developedusing recombinant Botulinum Neurotoxin Type A Light Chain (BoNT/A-LC)from List Biological Laboratories, Inc. (Campbell, Calif.) and laterconfirmed using Botulinum Neurotoxin Type A complex from BEI Resources(Manassas, Va.). Botulinum Type A Complex Toxoid (BoNT/A Complexinactivated by formalin) from Metabiologics, Inc (Madison, Wis.) alongwith heat-inactivated BoNT/A-LC (boiled for 30 min) were used to confirmthe specificity of the assay to BoNT/A activity in the studies. Studieswere performed using SNAPtide®, a peptide substrate labeled with theFITC/DABCYL FRET pair, purchased from List Biological Laboratories, Inc.(Campbell, Calif.). Later studies were performed using designed andsynthesized fluorescent peptide substrates (SEQ. ID NOS. 1 through 6)with the FAM/DABCYL FRET pair (Table 1). Hepes buffer solution andtris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich(Saint Louis, Mo.). Dithiothreitol (DTT), Triton X-100, zinc chloride(ZnCl₂), Tween® 20, Phosphate Buffered Saline (PBS) solution, and Bovineserum albumin (BSA) were purchased from Fisher Scientific (Waltham,Mass.).

TABLE 1 Synthesized fluorescent peptide substrates SEQ. ID NO. 1MDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSN(KDabcyl)TRIDEANQRATKML(K5Fam) SEQ. ID NO. 2MDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKAD SN(KDabcyl)TR E D IQATNRAKML(K5Fam) SEQ. ID NO. 3 MDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSN(KDabcyl)TR E D IQ N A R TA KML(K5Fam) SEQ. ID NO. 4MDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKAD SN(KDabcyl)TR E D IQ N AT A RKML(K5Fam) SEQ. ID NO. 5 MDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSN(KDabcyl)TR E D IQ N ATRA KML(K5Fam) SEQ. ID NO. 6(KDabcyl)TRIDEANQRATKML(K5Fam) where (KDabcyl) = Lys labeling by Dabcyl(or other quencher), (K5Fam) = Lys labeling by 5-Carboxyfluorescein (orother fluorophore), and QR = BoNT/A cleavage site Common PCR Inhibitors

Arizona road dust was purchased from Powder Technology, Inc.(Burnsville, Minn.). Arizona road dust was collected on a SASS® 3100filter cartridge; from Research International (Monroe, Wash.), andextracted into 1×PBS containing 0.05% (v/v) Triton X-100 using the SASS®3100 Dry Air Sampler and SASS® 3010 Particle Extractor systems fromResearch International. The extracted Arizona road dust was centrifugefor 5 minutes at 5000 rpm in an Eppendorf table top centrifuge, and thesupernatant was used for inhibition studies. Arizona road dust was alsoutilized for protease activity studies, where a 100 mg/mL Arizona roaddust mixture was made in H₂O, vortexed for 1 minute, then allowed tosettle for 30 minutes. The resulting supernatant was collected for usein the fluorescent peptide substrate based protease activityexperiments.

Diesel exhaust residue from a tractor was collected onto a SASS® 3100filter cartridge. The filter containing the exhaust residue wasextracted with 1×PBS containing 0.05% (v/v) Triton X-100 using the SASS®3010 Particle Extractor system. The resulting solution was used directlyin inhibition studies.

Humic acid was purchased from Fisher Scientific (Waltham, Mass.), andwas dissolved in 1×PBS containing 0.05% (v/v) Triton X-100 for use insubsequent inhibition studies.

BoNT/A Assay Development and Optimization in the Rotor-Gene® Q UsingSNAPtide®

Initial experiments tested a series of three SNAPtide® concentrations(5, 10, and 20 μM) and 6 amounts of BoNT/A-LC (0.28, 1.4, 2.8, 7.14,14.3, and 28.6 ng) in the following assay buffer: 50 mM Hepes buffer pH7.4+0.05% (v/v) Tween® 20. Reactions were incubated in the Rotor-Gene® Qreal-time PCR cycler using the following cycling profile: 60×1 minutecycles at 37° C. The Rotor-Gene® Q acquired a fluorescence signal at theend of each cycle and was able to detect BoNT/A-LC activity inreal-time. A 10 μM SNAPtide® was chosen as the optimal peptide substrateconcentration for the assay. Initial experiments in the Rotor-Gene® Qwere performed using 60×1 minute cycles, but later were adjusted to 55×1minute cycles to match the RAZOR® EX settings.

Next, different assay buffers were tested using the 10 μM SNAPtide®concentration and 3 amounts of BoNT/A-LC. The following buffers werecompared: 50 mM Hepes buffer pH 7.4+0.05% (v/v) Tween® 20, 50 mM Hepesbuffer pH 7.4+1 mg/mL BSA, 34.5 mM Hepes buffer pH 7.4+0.03% Tween20+0.31% PBS+0.02% (v/v) Triton X-100, and 34.5 mM Hepes buffer pH7.4+0.69 mg/mL BSA+0.31% PBS+0.02% (v/v) Triton X-100 (FIG. 1).PBS+Triton X100 is commonly used to extract agents, such as BoNT/A, fromcollected environmental samples. The assay was optimized for workingwith 0.31% PBS and 0.02% (v/v) Triton X-100. Tween® 20 and BSA arecommonly added to enhance protein stability during analysis. However,BSA caused negative controls containing 10 μM SNAPtide® and no BoNT/A-LCto fluoresce, so the use of Tween® 20 was preferred. As seen in FIG. 3,assays were incubated at 37° C. for 55 cycles (1 cycle/minute) withfluorescence acquisition at the end of each cycle. BSA caused thenegative controls to fluoresce, so the optimal assay buffer chosen was34.5 mM Hepes buffer pH 7.4+0.03% Tween® 20+0.31% PBS+0.02% TritonX-100. Thus, the Example moved forward using 34.5 mM Hepes buffer pH7.4+0.03% (v/v) Tween® 20+0.31% PBS+0.02% (v/v) Triton X-100 as theassay buffer.

Finally, the assay was optimized in the Rotor-Gene® Q to work with thefull length BoNT/A protein by testing different concentrations of ZnCl₂and DTT. The use of a reducing agent, such as DTT, is required to detectBoNT/A as the BoNT/A-LC and HC disulfide bonds needs to be broken toallow for BoNT/A-LC mediate peptide cleavage. The addition of ZnCl₂ tothe assay mix with DTT is necessary as DTT can chelate zinc, which isrequired for BoNT/A-LC protease activity. As seen in FIG. 4, theactivity of 1.9 ng and 7.5 ng BoNT/A-LC using varying concentrations ofZnCl₂ and DTT was tested. The addition of DTT alone reduced BoNT/A-LCactivity and 0.3 mM ZnCl₂ was found to be the optimal concentration ofZnCl₂ required to counteract this reduction in activity (data notshown). Addition of the common PCR inhibitors humic acid (0.16 μg/mL)and diesel exhaust residue (1:10 dilution) did not affect the activityof BoNT/A-LC in the presence of various DTT concentrations and 0.3 mMZnCl₂. In these experiments, 10 μM SNAPtide® and the following assaybuffer were used: 34.5 mM Hepes buffer pH 7.4+0.03% (v/v) Tween®20+0.31% PBS+0.02% (v/v) Triton X-100.

BoNT/A Assay Development and Optimization in the RAZOR® EX UsingSNAPtide®

The experiments performed in the Rotor-Gene® Q were translated to theRAZOR® EX. An initial experiment was performed in the RAZOR® EX using aconfiguration profile created specifically for this assay. Thisconfiguration profile used the following cycling parameters: 55×1 minutecycles at 37° C. while acquiring fluorescence at the end of each cycle.Referring to FIG. 5, a range of BoNT/A-LC amounts (0.11 ng-30 ng) wastested using 10 μM SNAPtide® and 50 mM Hepes pH 7.4+0.05% (v/v) Tween®20 assay buffer. The RAZOR® EX was able to detect BoNT/A-LC activity inreal-time. The ability of the RAZOR EX® was then tested to detectBoNT/A-LC activity using the 34.5 mM Hepes buffer pH 7.4+0.03% (v/v)Tween® 20+0.31% PBS+0.02% (v/v) Triton X-100 buffer. As seen in FIG. 6,a range of BoNT/A-LC (0.06 ng-7.5 ng)+10 μM SNAPtide® was assayed using34.5 mM Hepes buffer pH 7.4+0.03% (v/v) Tween 20+0.31% PBS+0.02% (v/v)Triton X-100 buffer. The RAZOR® EX was able to detect BoNT/A-LC activityin real-time.

In order to optimize the ZnCl₂ and DTT concentrations for use in theRAZOR® EX, the assay buffer was altered to 32.2 mM Hepes buffer pH7.4+0.03% Tween 20+0.30% PBS+0.02% Triton X-100, and used 9.64 μMSNAPtide®. Referring to FIG. 7, the combination of 2.5 mM DTT and 0.3 mMZnC12 was compatible, as the addition of DTT is required to detect thefull length BoNT/A protein.

The 2.5 mM DTT and 0.3 mM ZnCl₂ conditions were chosen as optimalconcentrations and therefore utilized to test the performance of theassay in the RAZOR® EX in the presence of common PCR inhibitors, as seenin FIGS. 8-10. As seen in FIG. 8, 1.9 ng-7.5 ng BoNT/A-LC in thepresence of diesel exhaust residue established that diesel exhaustresidue did not inhibit BoNT/A-LC activity. As seen in FIG. 9, 0.04mg/mL-0.08 mg/mL of Arizona road dust did not inhibit 7.5 ng BoNT/A-LC.Finally, as seen in FIG. 10, 7.5 ng BoNT/A-LC established that theBoNT/A-LC assay is compatible with humic acid concentrations up to 0.23μg/mL.

Design of SEQ. ID NO. 1 and SEQ. ID NO. 2 BoNT/A Fluorescent PeptideSubstrates

Based on the studies utilizing SNAPtide® in the Rotor-Gene® Q and theRAZOR® EX, the detection of biologically active BoNT/A-LC protein toxinwas shown to be possible on a qPCR platform given the correct bufferconditions and PCR parameters. While SNAPtide® is a good candidate forBoNT/A-LC activity, it did have issues with stability, solubility, anddetection signal. The instability of the SNAPtide® peptide was shown ina number of experiments where the negative control samples showed largeand unexpected increases in fluorescence, followed by drops influorescence signal overtime. Additionally, solubility issues wereidentified in some of our solutions where precipitation of SNAPtide® wasobserved in both stock samples and experimental samples. Finally, thedetection signal of SNAPtide® was not ideal as a large amount of BoNT-ALC is required in order to generate a high enough signal over noise toallow detection of BoNT-A LC activity based on an algorithmicdetermination. Therefore, a new BoNT-A peptide substrate design wasutilized.

The present invention includes six fluorescent peptide substrates forBoNT/A detection (Table 1). The SNAPtide® peptide substrate is known toonly include a short SEQ. ID NO. (about 15 amino acids in length)resembling the BoNT/A cleavage site on SNAP-25. While BoNT/A can cleavethe SNAPtide® peptide substrate, BoNT/A binding to SNAPtide® is notideal due to the exclusion of BoNT/A binding domains found on SNAP-25.Therefore, to increase the binding efficiency of BoNT/A, a 59 amino acidfluorescent peptide (SEQ. ID NO. 1) based on the SNAP-25/BoNT/Ainteraction was designed. Shown in the three-dimensional Swiss PDBviewer representations in FIG. 11, BoNT/A-LC (in red) interacts with theC-terminal portion (Green/Blue) of the SNAP-25 protein. The BoNT/Acleavage area (dark blue) and cleavage site (light blue) on SNAP-25 fallwithin a pocket within BoNT/A-LC protease (FIG. 11A-B). Due to theacceptable distance (32.8 Angstroms) between the lysines located on thepeptide SEQ. ID NO. (FIG. 11B), these residues were chosen as thelocation of fluorophore and quencher for the FRET pair. While theinternal region of the peptide was not shown to have any obviousinteractions with the BoNT/A-LC (FIG. 11C), an alpha-helical structurelocated in a SNARE region of the SNAP-25 did interact with BoNT/A-LC(FIG. 11D). Based on these observations, the SEQ. ID NO. 1 fluorescentpeptide was designed (Table 1).

To design a negative control fluorescent peptide, it had to meet twocriteria: 1) Cannot be cleaved to BoNT/A-LC and 2) Must be sensitive toany interferents that affect the SEQ. ID NO. 1 peptide. Based on thesecriteria, four potential negative control fluorescent peptides weredesigned (SEQ. ID NOS. 2-5; Table 1). In each of the peptides, thefluor/quencher region was mutated in such a way to destroy the BoNT/Arecognition/cleavage site. These SEQ. ID NO. s were then run through theExPASy cleavage predictor software to determine if the fluor/quencherregions still possessed similar cleavage maps based on the knownproteases (FIG. 12). While SEQ. ID NO. 1 can be cleaved by 16 differentproteases in 36 different potential cleavages, SEQ. ID NO. 2 was shownto be cleaved by the same number of proteases in the same number ofcleavages (FIG. 12A). Additionally, the SEQ. ID NO. 2 fluor/quenchercontained an 8 amino acid difference in the peptides BoNT/A cleavagerecognition site compared to SEQ. ID NO. 1 (Table 1). Therefore, theSEQ. ID NO. 2 negative control/interferents sensing peptide was chosenover the other negative controls due to various reasons as listed in theFIGS. 12B-C.

BoNT/A Assay Development and Optimization in the FilterMax® F5 UsingSEQ. ID NO. 1

Initial experiments with SEQ. ID NO. 1 were performed to determine itscapability in detecting BoNT/A-LC activity compared to theproof-of-principle peptide SNAPtide®. The FilterMax® F5 fluorimeter wasused in this testing because it possesses a heat controlled sampledetection area (can be regulated from 25 to 45° C.) and can preciselydetect changes in fluorescence of a broad detection range over a timecourse. Similar concentrations of SEQ. ID NO. 1 and SNAPtide® (10 μM)were used to detect 3 different amounts of BoNT/A-LC (7.5, 15, and 30ng) in a 96 well plate (100 μL per well). Similar buffer conditions (30mM Hepes pH 7.4, 0.2% Tween 20), that were optimized from theproof-of-principle studies, were used for all the samples in the assay.The reactions were incubated at 37° C. for 60 minutes, with fluorescencereadings taken at 1 minute intervals. As seen in FIG. 13, a greaterincrease in fluorescence overtime was observed in all SEQ. ID NO. 1samples compare to SNAPtide® samples at all BoNT/A-LC conditions tested.These results show that the SEQ. ID NO. 1 fluorescence substrateproduces a greater signal than that of SNAPtide®. Additionally, based onobservational studies SEQ. ID NO. 1 is more stable (does notprecipitate) and produces very little fluorescence background noisecompared to SNAPtide®.

Importance of the BoNT/A Binding Region in SEQ. ID NO. 1 Compared toSEQ. ID NO. 6

As SEQ. ID NO. 1 was designed to possess an alpha-helical binding regionto enhance its interaction with BoNT/A (FIG. 11D), the next test wasdesigned to assess the importance of this region in detection BoNT/Aactivity. SEQ. ID NO. 6 was designed to lack the BoNT/A binding regionthat is found in SEQ. ID NO. 1 (Table 1). Similar concentrations of SEQ.ID NO. 1 and SEQ. ID NO. 6 (10 μM) were used to detect 2 differentamounts of BoNT/A-LC (15 and 30 ng) in a 96 well plate (100 μL per well;30 mM Hepes pH 7.4, 0.2% Tween 20) to be analyzed on the FilterMax® F5fluorimeter. The reactions were incubated at 37° C. for 60 minutes, withfluorescence readings taken at 1 minute intervals. As seen in FIG. 14,while SEQ. ID NO. 1 was able to detect BoNT/A-LC, the lack of the BoNT/Abinding region on SEQ. ID NO. 6 rendered it incapable of detectingBoNT/A-LC activity.

SEQ. ID NO. 1 Detects Biologically Active BoNT/A, but not Heat orFormalin Inactivated BoNT/A

The present invention was designed to produce an assay capable ofdetecting only the biologically active form of BoNT/A. Therefore,versions of BoNT/A that were known to be inactivated either throughheating (heat inactivated BoNT/A-LC) or through chemical treatment withformalin (BoNT/A Toxoid) were assessed in our studies with SEQ. IDNO. 1. Due to the high signal produced using SEQ. ID NO. 1, we were ableto optimize our conditions to only use 1 μM instead of 10 μM SEQ. ID NO.1 (data not shown). SEQ. ID NO. 1 (1 μM) was used in a detection assaywith BoNT/A-LC (1 and 5 ng), heat inactivated BoNT/A-LC (5 and 80 ng),and BoNT/A Toxoid (20 and 200 ng) in a 96 well plate (100 μL per well;30 mM Hepes pH 7.4, 0.2% Tween 20) to be analyzed on the FilterMax® F5fluorimeter. The reactions were incubated at 37° C. for 60 minutes, withfluorescence readings taken at 1 minute intervals. As seen in FIG. 15A,SEQ. ID NO. 1 was able to detection biologically active BoNT/A-LC (5ng), but not heat inactivated BoNT/A-LC, even when present at highconcentrations. Similarly, as seen in FIG. 15B, SEQ. ID NO. 1 was ableto detect biologically active BoNT/A-LC (1 ng), but not BoNT/A Toxoid,even when present at high concentrations. Therefore, assays utilizingSEQ. ID NO. 1 can differentiate between biologically active andbiologically inactive BoNT/A.

Development of SEQ. ID NO. 2 as a Negative Control/Interferent Sensorfor the BoNT/A Assay

The present invention was designed to produce a BoNT/A detection assaycapable of producing a signal that can be inputted into an algorithm todetermine if a sample possesses or lacks biologically active BoNT/A. Inorder to make this determination, the algorithm not only needs a signalfrom an input that can detect the presence or absence of BoNT/A, but italso requires a signal that is a negative control, which providesinformation regarding the background fluorescence in a system. Thisnegative control must be insensitive to BoNT/A mediated cleavage, yet itmust be able to register the background noise (interference) present inthe system which may affect the detecting substrate (SEQ. ID NO. 1).Therefore, SEQ. ID NO. 2 was designed as this negativecontrol/interference sensor. SEQ. ID NO. 1 and SEQ. ID NO. 2 (1 μM) wereused in a detection assay with BoNT/A-LC (5 and 50 ng) in a 96 wellplate (100 μL per well; 30 mM Hepes pH 7.4, 0.2% Tween 20) to beanalyzed on the FilterMax® F5 fluorimeter. The reactions were incubatedat 37° C. for 60 minutes, with fluorescence readings taken at 1 minuteintervals. As seen in FIG. 16A, while SEQ. ID NO. 1 was able to detectbiologically active BoNT/A-LC (5 and 50 ng), SEQ. ID NO. 2 showed noincrease in fluorescence over time in the presence of BoNT/A-LC, even athigh concentrations. Therefore, SEQ. ID NO. 2 is capable of acting as anegative control in the BoNT/A detection assay. In a separate test, SEQ.ID NO. 1 and SEQ. ID NO. 2 (1 μM) were used in a detection assay with100 mg/mL Arizona road dust (ARD), a common PCR inhibitor, with observedprotease activity. This assay was performed in a 96 well plate (100 μLper well; 30 mM Hepes pH 7.4, 0.2% Tween 20) and analyzed on theFilterMax® F5 fluorimeter at 37° C. for 60 minutes, with fluorescencereadings taken at 1 minute intervals. As seen in FIG. 16B, ARDnon-specifically caused an increase in fluorescence (due to the presenceof proteases within the ARD) in SEQ. ID NO. 1 over the time course ofthe assay. Similar to SEQ. ID NO. 1, ARD also caused an increase influorescence over time in SEQ. ID NO. 2. Therefore, SEQ. ID NO. 2 can beutilized as a negative control/interferents sensor for the BoNT/Adetection assay.

SEQ. ID NO. 1 Detects BoNT/A Activity in the Presence of Common PCRInhibitors

To determine if common PCR inhibitors affect the ability of SEQ. ID NO.1 to detect the activity of BoNT/A-LC in the presence of common PCRinhibitors, diesel exhaust (FIG. 17) and humic acid (FIG. 18) weretested. SEQ. ID NO. 1 (1 μM) was incubated with BoNT/A-LC (2.5 and 5 ng)in the presence of non-diluted diesel exhaust (30% assay volume) orhumic acid (250 ng/mL) in a 96 well plate (100 μL per well; 30 mM HepespH 7.4, 0.2% Tween 20) and analyzed on the FilterMax® F5 fluorimeter at37° C. for 60 minutes, with fluorescence readings taken at 1 minuteintervals. As seen in FIGS. 17 and 18, while common PCR inhibitorsslightly affect the overall fluorescence in the BoNT/A detection assay,small amounts of BoNT/A-LC were still capable of generating a largeincrease in fluorescence signal overtime. Therefore, SEQ. ID NO. 1 candetect BoNT/A-LC activity in the presence of common PCR inhibitors.

BoNT/A Assay Reducing Agent Optimization Using TCEP and DTT

Next, the assay was optimized to work with the full length BoNT/A(containing both heavy and light chains). Initially, the assay wasoptimized to work with 0.3 mM ZnCl₂ and the 2.5 mM DTT reducing agent.However, TCEP, a reducing agent more stable than DTT and that does notchelate zinc, was tested in the BoNT/A assay. SEQ. ID NO. 1 (1 μM) wasused in a detection assay with BoNT/A-LC (5 ng) either with DTT (2.5 mM,with 0.3 mM ZnCl₂), TCEP (2.5 mM) or no reducing agent, in a 96 wellplate (100 μL per well; 30 mM Hepes pH 7.4, 0.2% Tween 20). Thereactions were incubated at 37° C. for 60 minutes in the FilterMax® F5fluorimeter, with fluorescence readings taken at 1 minute intervals. Asseen in FIG. 19, samples with 2.5 mM TCEP performed better than sampleswith DTT (and zinc), and even better than samples containing no reducingagent. Therefore, TCEP was chosen as the reducing agent used in theBoNT/A activity detection assay.

Detection of Full BoNT/A Holotoxin Complex in the BoNT/A Assay with TCEP

To determine the optimal TCEP concentration required for BoNT/AHolotoxin detection, SEQ. ID NO. 1 (1 μM) was used in a detection assaywith BoNT/A Holotoxin (5 ng) either with or without TCEP (1.0 and 2.0mM), in a 96 well plate (100 μL per well; 30 mM Hepes pH 7.4, 0.2% Tween20). The reactions were incubated at 37° C. for 60 minutes in theFilterMax® F5 fluorimeter, with fluorescence readings taken at 1 minuteintervals. As seen in FIG. 20, samples with 1.0 mM TCEP performed betterthan samples with 2.0 mM TCEP, noted by the greater increase influorescence over time. As expected, samples that did not contain TCEPwere unable to detect BoNT/A Holotoxin. Therefore, 1.0 mM TCEP waschosen as the reducing agent concentration used in the BoNT/A activitydetection assay.

BoNT/A Assay Development and Optimization in the RAZOR® EX Using SEQ. IDNO. 1 and 2

One embodiment of the present invention is an assay to detectbiologically active BoNT/A on a qPCR platform. The previous bufferoptimizations and designed fluorescence peptide substrates which wereoptimized on the FilterMax® F5 fluorimeter can be translated to performon a qPCR platform. To test this, the RAZOR® EX qPCR protocol wasoptimized (based on information gathered from assays performed on theRAZOR® EX with SNAPtide®). The optimizations consisted of making anisothermal cycling protocol to be run at 37° C., adapting the read timesto be performed at 1 minute intervals, and collecting PCR run data asraw data for subsequent graphing and algorithm processing. As seen inFIG. 21, SEQ. ID NO. 1 and SEQ. ID NO. 2 (both at 1 μM) were used in adetection assay with BoNT/A-LC (7.5 ng) in a 6×2 pouch (200 μL per well;30 mM Hepes pH 7.4, 0.2% Tween 20, 1.0 mM TCEP). The reactions wereincubated at 37° C. for 45 minutes in the RAZOR® EX, with fluorescenceread at 1 minute intervals. While this assay was for a total of 45minutes, such a strong positive signal was generated against a lowbackground signal that a positive BoNT/A determination based onalgorithmic determination and weighting (based on overall fluorescenceincrease and increase in fluorescence slope) could be made withinapproximately 10 minutes (10 cycles).

BoNT/A Assay Development and Optimization in the Genedrive® Using SEQ.ID NO. 1 at 42° C.

To further test the BoNT/A assay, we utilized the Genedrive® portableqPCR system. To test this assay, we first optimized the Genedrive® qPCRprotocol making an isothermal cycling protocol to be run at 42° C.(standardized on this machine), adapting the read times to be performedat 1 second intervals, and collecting PCR run data as raw data forsubsequent graphing and algorithm processing. As seen in FIG. 22, SEQ.ID NO. 1 (1 μM) was used in a detection assay with BoNT/A-LC (1.0 and5.0 ng) in a 3×1 Genedrive® cassette (20 μL per well; 30 mM Hepes pH7.4, 0.2% Tween 20, 1.0 mM TCEP). The reactions were incubated at 42° C.for 60 minutes in the Genedrive®, with fluorescence readings taken at 1second intervals. While this assay was run for a total of 60 minutes,such a strong positive signal was generated against a background signalthat a positive BoNT/A determination based on algorithmic determinationand weighting (based on overall fluorescence increase and increase influorescence slope) could be made within approximately 15 minutes.Additionally, these results show that the BoNT/A assay can be run at 42°C., as well at 37° C.

BoNT/A Assay Limits of Detection Using SEQ. ID NO. 1

To determine the limit of detection of biologically active BoNT/A LCusing SEQ. ID NO. 1, SEQ. ID NO. 1 (1 μM) was incubated with varyingamounts of BoNT/A-LC (0.008, 0.04, and 0.2 ng) in a 96 well plate (100μL per well; 30 mM Hepes pH 7.4, 0.2% Tween 20, 1.0 mM TCEP) andanalyzed on the FilterMax® F5 fluorimeter at 37° C. for 60 minutes, withfluorescence readings taken at 1 minute intervals. As seen in FIG. 23,the limit of the detection for the SEQ. ID NO. 1 BoNT/A activity assaywas at least 0.04 ng BoNT/A-LC over a 60 minute time period. Longerincubations may increase this limit of detection.

Development and Optimization of a Toxin Algorithm for the Detection ofBoNT/A

The algorithm according to the present invention was developed to allowthe presence or absence determination of biologically active toxin on aqPCR platform. The detection algorithm that is typically included inqPCR fluorescence detection instruments (e.g. Rotor-Gene® Q, RAZOR® EX,Genedrive®) is optimized to interpret fluorescence data from individualgenetic samples that generate exponential increases in fluorescence whena critical threshold (CT) point is achieved. Genetic samples are “calledpositive” by a qPCR instrument's algorithm based on this exponentialfluorescence increase (graphically displayed as a sigmoidal curve; cyclevs. fluorescence), which can be distinguished from negative samples thatdo not generate exponential fluorescence increases (graphicallydisplayed as a flat straight line). Contrary to genetic samples, toxinsamples generate non-exponential increases in fluorescence (graphicallydisplayed as a straight line with a positive slope; time vs.fluorescence). Additionally, toxin samples require the comparison of theunknown samples (could be positive or negative) to a control sample (anegative sample) to determine a signal to noise ratio. Therefore, a qPCRinstrument's genetic algorithm, which inherently lacks the capability tofactor in a signal to noise ratio and is weighted to distinguishexponential data from non-exponential data, is not useful for datagenerated from toxin assays.

The first approach that was applied to the data from the initialSNAPtide experiments was to compare the fluorescence at the end of eachcycle to a threshold value. The concept was that biologically activetoxin was present in the sample if a specified threshold was exceeded;otherwise, toxin was absent from the sample. Observations of the resultsfrom this initial approach (data not shown) showed that this method wasunreliable as background noise could exceed the threshold under certainconditions (e.g., interferents). These observations helped to illustratethe importance of using a negative control for determining the level ofbackground fluorescence.

Development of the algorithm began by importing the raw data outputsinto Microsoft Excel for 152 sample sets run on the FilterMax® F5fluorimeter and 15 sample sets run on the RAZOR® EX. The purpose of thiswas to chart the data for comparison and performing investigativecalculations to identify trends and conditions of significance. Thefollowing progression was applied to each of these 167 sample sets.First, the raw fluorescence values were graphed, as seen in FIG. 25, forboth the assay and the negative control. This graph illustrated that thetwo lines diverged, but also experienced marked variability betweenconsecutive cycles. Next, the average fluorescence of the negativecontrol was graphed, as seen in FIG. 26, to smooth out the variabilityof the unprocessed data. The background/negative control data valueswere then subtracted from the assay data values and graphed, as seen inFIG. 26, for comparison. This illustrated that the backgroundfluorescence could vary somewhat depending on which well or lane thesample was tested in because some of the subtractions resulted in afluorescence value less than zero for the assay, which is not an actualpossible condition.

The divergence of assay fluorescence values from the negative controlfluorescence was identified as an important contributor to determiningthe presence of a biologically active toxin, but it was also identifiedthat the rate at which these values diverged (or the slope of the curve)was also important. To study this, graphs of the slopes at each cycle,as seen in FIG. 27, and the change in the slope over time, as seen inFIG. 28, were created. These graphs showed that it could be difficult tocompare the slope of the background fluorescence curve with the slope ofthe assay curve at each cycle with a good degree of accuracy. Thecumulative slope, as seen in FIG. 29, and the deltas between cycles ofthe cumulative slope, as seen FIG. 30, were then graphed, whichdemonstrate that there is a higher variability in the first 10 cycles ofa run. This finding lead to the conclusion that we needed an algorithmthat could even out the high variability over time. Comparisons of thediscrete and cumulative slopes for unprocessed values, as see in FIGS.31 and 32, and background adjusted values, as seen in FIGS. 33 and 34,were graphed to reinforce the idea that the algorithm should be using anaveraged slope value instead of the slope at a specified point in time.

Analysis of the standard deviations of fluorescence values for theassay, negative control, and the averages of these showed that there isa more noticeable increase over time for the assay than for the negativecontrol, as seen in FIG. 35. This finding started on the path to usingstandard deviation as the preferred unit of measure for determining ifbiologically active toxin is present in a sample. Next, the standarddeviations of the discrete sloped, as seen in FIG. 36, and cumulativesloped, as seen in FIG. 37, were compared. This confirmed findings fromprevious comparisons that cumulative slope is a better indicator of thepresence of biologically active toxin and that standard deviation is anacceptable unit of measure to use for this determination. All of thesefindings were used as inputs to developing the toxin detectionalgorithm.

In order to implement the botulinum assay of the present invention in alegacy or conventional real-time PCR detector that is adapted strictlyfor real-time PCR routines and assays, the present invention alsoencompasses a detection algorithm to identify the presence ofbiologically active toxin that is instrument independent. The algorithmof the present invention is intended to be applied periodically at theend of each time cycle during an assay, and the output from thealgorithm becomes more reliable as the number of completed cyclesincreases. The contributing factors to the algorithm are thefluorescence values as measured by the instrument of choice and thechange in fluorescence over time. The algorithm requires a negativecontrol assay sample to be run simultaneously on the same instrument,which provides a measure of the background fluorescence in the samplenot resulting from peptide cleavage activity by a toxin (e.g., BoNT/A).

More specifically, the algorithm determines if the fluorescence and thechange over time in fluorescence for the assay differ significantlyenough from the negative control background fluorescence and the changein background fluorescence to indicate peptide cleavage activity. Themagnitude of the delta from the background values is positivelycorrelated to the likelihood of the presence of biologically activetoxin. The larger the differences for each of the factors, the higherthe likelihood that BoNT/A is present in the sample.

The algorithm relies on fluorescence values that have been obtained forall completed time cycles. First, the standard deviation and averagefluorescence are calculated for the negative control fluorescencevalues. The average fluorescence is also calculated from the samplecontrol values. Using these calculated values, the algorithm determinesthe number of positive standard deviations (up to 10) between thenegative control average fluorescence and the sample averagefluorescence. (A sample average fluorescence lower than the negativecontrol average fluorescence is assigned a resulting value of 0 standarddeviations.) The resulting number is then weighted by the fluorescenceweight factor to obtain the fluorescence sub-score for that cycle. Afluorescence weight factor is used to assign the level of importancethat fluorescence contributes to the determination of the presence orabsence of a biologically active toxin. The fluorescence sub-scores areadded together to achieve the overall fluorescence sub-score.

Next, the change in fluorescence (slope) between consecutive cycles isevaluated. The standard deviation and average slope are calculated forthe negative control fluorescence values. The average slope is alsocalculated from the sample control values. Using these calculatedvalues, the algorithm determines the number of positive standarddeviations (up to 10) between the negative control average slope and thesample average slope. (A sample average slope lower than the negativecontrol average slope is assigned a resulting value of 0 standarddeviations.) The resulting number is then weighted by the slope weightfactor to obtain the slope sub-score for that cycle. A slope weightfactor is used to assign the level of importance that slope contributesto the determination of the presence or absence of a biologically activetoxin. The slope sub-scores are added together to achieve the overallslope sub-score.

The overall fluorescence sub-score and the overall slope sub-score arethen added together and then divided by the maximum possible points toarrive at the likelihood score. This score can then be compared to oneor more thresholds to provide a ranking of the presence of biologicallyactive toxin. For instance, comparing the score against a singlethreshold will indicate positive vs. negative. Similarly, comparing thescore against three thresholds or ranges will provide low, medium, highindications of a positive presence of the biologically active toxin.

EXAMPLE 2

The mathematical formulas and variables for determining each of thesteps of the algorithm may be seen in FIG. 24. FIGS. 25-37 are graphs ofthe results of the steps of the algorithm of the present invention asapplied to the exemplary raw test data of Table 2 below:

TABLE 2 Raw Test Data 0:00:00 1 17219782 16639028 16929405 1694125016127615 16534432.5 0:01:03 2 16954224 16570812 16762518 1689820016291588 16594894 0:02:06 3 17348886 16617691 16983288.5 1706409616192753 16628424.5 0:03:09 4 17037452 16571507 16804479.5 1686847016139195 16503832.5 0:04:12 5 17063478 16493083 16778280.5 1697495616113786 16544371 0:05:15 6 16995928 16555440 16775684 16897506 1610226216499884 0:06:18 7 16997406 16522870 16760138 16928310 16102754 165155320:07:21 8 17049742 16419397 16734569.5 16958966 16182215 16570590.50:08:24 9 17187260 16568604 16877932 16991158 16241493 16616325.50:09:27 10 17127746 16612530 16870138 17091148 16158359 16624753.50:10:29 11 17489694 16721705 17105699.5 17135356 16393144 167642500:11:32 12 17073140 16455406 16764273 17003150 16292940 16648045 0:12:3513 17162860 16651130 16906995 17032674 16263690 16648182 0:13:38 1417180334 16635167 16907750.5 17168432 16399479 16783955.5 0:14:41 1517290586 16598597 16944591.5 17113964 16396502 16755233 0:15:44 1617302706 16724791 17013748.5 17235722 16379507 16807614.5 0:16:47 1717419846 16856302 17138074 17346484 16612662 16979573 0:17:50 1817548768 16974448 17261608 17374476 16559293 16966884.5 0:18:53 1917431150 16769061 17100105.5 17402868 16559305 16981086.5 0:19:56 2017525446 16787790 17156618 17441596 16634751 17038173.5 0:20:59 2117443736 16832370 17138053 17433534 16665144 17049339 0:22:02 2217502950 16827952 17165451 17464344 16838100 17151222 0:23:05 2317767910 17002642 17385276 17661374 16788834 17225104 0:24:08 2417409266 16875390 17142328 17625426 16799602 17212514 0:25:11 2517439062 16791298 17115180 17603460 16859024 17231242 0:26:14 2617844372 16958290 17401331 17690426 16814306 17252366 0:27:17 2717522334 16931420 17226877 17606234 16768304 17187269 0:28:20 2817472412 16915442 17193927 17705496 16960658 17333077 0:29:23 2917535798 17028778 17282288 17857644 17060324 17458984 0:30:26 3017630158 17067400 17348779 17948812 17170650 17559731 0:31:29 3117606646 16984718 17295682 17762786 17053586 17408186 0:32:32 3217582174 16880812 17231493 18037586 17240350 17638968 0:33:34 3317613928 17060662 17337295 17886246 17218370 17552308 0:34:37 3417564772 17119882 17342327 17999864 17198184 17599024 0:35:40 3517985124 17108548 17546836 18146340 17307620 17726980 0:36:43 3617647100 17003370 17325235 18020932 17235566 17628249 0:37:46 3717621018 17046734 17333876 18041022 17375910 17708466 0:38:49 3817636718 17009510 17323114 17949748 17263460 17606604 0:39:52 3917625832 16817450 17221641 17960704 17244136 17602420 0:40:55 4017970444 17141602 17556023 18258588 17547610 17903099 0:41:58 4117670898 17016084 17343491 18172040 17581468 17876754 0:43:01 4217707308 17035982 17371645 18183054 17457358 17820206 0:44:04 4317683410 17089802 17386606 18330410 17610868 17970639 0:45:07 4417754202 17058480 17406341 18275842 17577324 17926583 0:46:10 4517781308 17112792 17447050 18463180 17673186 18068183 0:47:13 4617724302 17110662 17417482 18385432 17806612 18096022 0:48:16 4717716832 17185570 17451201 18513974 17789102 18151538 0:49:19 4817733516 17043026 17388271 18561598 17705960 18133779 0:50:22 4917684590 17028736 17356663 18436202 17776382 18106292 0:51:24 5017610388 17012552 17311470 18454758 17701072 18077915 0:52:27 5117635742 17042302 17339022 18607506 17856482 18231994 0:53:30 5217937962 17134502 17536232 18486846 17739870 18113358 0:54:33 5317711400 17081744 17396572 18525500 17660466 18092983 0:55:36 5417746340 17135416 17440878 18654216 17912552 18283384 0:56:39 5518025748 17261254 17643501 18837796 18026790 18432293 0:57:42 5617837154 17217404 17527279 18908546 18053532 18481039 0:58:45 5717759734 17116800 17438267 18795458 18018540 18406999 0:59:48 5817751840 17179348 17465594 18852418 18077902 18465160 1:00:51 5917834238 17192950 17513594 18839772 18060456 18450114 1:01:54 6017781560 17167442 17474501 18879812 18142536 18511174 1:02:57 6117897182 17219990 17558586 18978560 18162694 18570627

It should be recognized by those of skill in the art that the algorithmof the present invention may be programmed into or as part of theoperating system of a device, such as a real-time PCR detection system,to enable the detection algorithm to be applied to a target sample. Forexample, the algorithm may physically incorporated into a PCR instrumentwill depend on the PCR instrument and vendor cooperation, and could bedone by implementing the algorithm in code modules added to theinstrument. Alternatively, the algorithm may be implemented on a laptopconnected to the PCR instrument to analyze the data if it is unable tobe incorporated directly into the PCR instrument

What is claimed is:
 1. A device for detecting the presence ofbiologically active botulinum toxin, comprising: a chamber for receivinga test sample, said chamber including a peptide substrate comprisingSEQ. ID NO. 1 that is labeled with a fluorophore and a quencher and hasa cleavage site responsive to biologically active botulinum toxinpositioned between said fluorophore and said quencher; a detector formeasuring the amount of fluorescence emitted from said sample andoutputting a corresponding fluorescence reading positioned proximatelyto said chamber; a microcontroller interconnected to said detector thatis programmed to acquire said fluorescence reading and to determinewhether any biologically active botulinum toxin is present in saidsample based on said fluorescence reading.
 2. The device of claim 1,wherein said chamber comprises a real-time PCR detector and saidmicrocontroller is associated with said PCR detector and has beenadditionally programmed to acquire said fluorescence reading after aplurality of cycles and to determine whether any biologically activebotulinum toxin is present in said sample based on the change in saidfluorescence reading over a plurality of said cycles.
 3. The device ofclaim 1, further comprising a negative control sample positioned in saidchamber that has fluorescence that is separately detectable by saiddetector.
 4. A device for detecting the presence of biologically activebotulinum toxin, comprising: a chamber for receiving a test sample, saidchamber including a peptide substrate labeled with a fluorophore and aquencher and having a cleavage site responsive to biologically activebotulinum toxin positioned between said fluorophore and said quencher; adetector for measuring the amount of fluorescence emitted from saidsample and outputting a corresponding fluorescence reading positionedproximately to said chamber; a microcontroller interconnected to saiddetector that is programmed to acquire said fluorescence reading and todetermine whether any biologically active botulinum toxin is present insaid sample based on said fluorescence reading; and a negative controlsample positioned in said chamber that has fluorescence that isseparately detectable by said detector, wherein said negative controlsample is a peptide selected from the group consisting of SEQ. ID NO. 2,SEQ. ID NO. 3, SEQ. ID NO. 4, and SEQ. ID NO.
 5. 5. The device of claim3, wherein said microcontroller is programmed to determine whetherbiologically active botulinum toxin in present in said sample based onthe change in magnitude of said fluorescence reading of said test sampleover a plurality of said cycles relative to the change in magnitude ofsaid fluorescence reading of said negative control sample over saidplurality of said cycles.
 6. The device of claim 5, wherein saidmicrocontroller is further programmed to determine whether biologicallyactive botulinum toxin in present in said sample based on an averagechange in slope of said fluorescence of said negative control sampleover said plurality of said cycles and an average change in slope ofsaid fluorescence of said test sample over said plurality of saidcycles.
 7. The device of claim 4, wherein said negative control sampleis a peptide selected from the group consisting of SEQ. ID NO. 2, SEQ.ID NO. 3, SEQ. ID NO. 4, and SEQ. ID NO. 5.